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BMC Evolutionary Biology Aug 2014Lysozyme g is an antibacterial enzyme that was first found in the eggs of some birds, but recently has been found in additional species, including non-vertebrates. Some...
BACKGROUND
Lysozyme g is an antibacterial enzyme that was first found in the eggs of some birds, but recently has been found in additional species, including non-vertebrates. Some previously characterized lysozyme g sequences are suggested to have altered secretion potential and enzymatic activity, however the distribution of these altered sequences is unknown. Duplicated copies of the lysozyme g gene exist in some species; however, the origins of the duplicates and their roles in altered function are unclear.
RESULTS
We identified 234 lysozyme g sequences from 118 vertebrate species, including 181 sequences that are full or near full length representing all vertebrate classes except cartilaginous fish. Phylogenetic analysis shows that most lysozyme g gene duplicates are recent or lineage specific events, however three amplification events are more ancient, those in an early amniote, an early mammal, and an early teleost. The older gene duplications are associated with changes in function, including changes in secretion potential and muramidase antibacterial enzymatic activity.
CONCLUSIONS
Lysozyme g is an essential muramidase enzyme that is widespread in vertebrates. Duplication of the lysozyme g gene, and the retention of non-secreted isozymes that have lost enzymatic activity indicate that lysozyme g has an activity other than the muramidase activity associated with being an antibacterial enzyme.
Topics: Animals; Base Sequence; Evolution, Molecular; Geese; Gene Duplication; Multigene Family; Muramidase; Phylogeny
PubMed: 25167808
DOI: 10.1186/s12862-014-0188-x -
The Journal of Physical Chemistry. B Feb 2022The interaction between graphene oxide (GO) and lysozyme (LYZ) in aqueous solution was investigated for GO specific surface area determination and for the thermodynamic...
The interaction between graphene oxide (GO) and lysozyme (LYZ) in aqueous solution was investigated for GO specific surface area determination and for the thermodynamic description of the process. It was experimentally proved that LYZ is a much better adsorbate than the most common methylene blue, allowing the determination of genuine GO surface area. Our fluorescence spectroscopy results indicate that LYZ molecules interact with GO at high- and low-affinity sites depending on the surface coverage, reflecting the protein mono- and multilayer formation, respectively. The lack of the secondary structure changes confirms LYZ usability as a model adsorbate. The calculated values of thermodynamic parameters (Δ(Δ) = -195.0 kJ/mol and Δ(Δ) = -621.3 J/molK) indicate that the interactions are exothermic, enthalpy-driven. All the reported results reveal the physical nature of the LYZ-GO interaction at the studied concentration ratios.
Topics: Adsorption; Graphite; Muramidase; Thermodynamics
PubMed: 35077166
DOI: 10.1021/acs.jpcb.1c08294 -
Nature Communications Mar 2020We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states....
We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.
Topics: Bacteriophage T4; Biocatalysis; Crystallography, X-Ray; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; Monte Carlo Method; Muramidase; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Viral Proteins
PubMed: 32144241
DOI: 10.1038/s41467-020-14886-w -
Protein Science : a Publication of the... Oct 2018A simple "diffusion-to-capture" model is used to estimate the upper limit to the growth rate of macromolecular crystals under conditions when the rate limiting process...
A simple "diffusion-to-capture" model is used to estimate the upper limit to the growth rate of macromolecular crystals under conditions when the rate limiting process is the mass transfer of sample from solution to the crystal. Under diffusion-limited crystal growth conditions, this model predicts that the cross-sectional area of a crystal will increase linearly with time; this prediction is validated by monitoring the growth rate of lysozyme crystals. A consequence of this analysis is that when crystal growth is diffusion-limited, micron-sized crystals can be produced in ~1 s, which would be compatible with the turnover time of many enzymes. Consequently, the ability to record diffraction patterns from sub-micron sized crystals by X-ray Free Electron Lasers and micro-electron diffraction technologies opens the possibility of trapping intermediate enzyme states by crystallization.
Topics: Crystallization; Humans; Macromolecular Substances; Muramidase
PubMed: 30056633
DOI: 10.1002/pro.3491 -
The Journal of Chemical Physics Sep 2013Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or...
Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the entire cascade of aggregation intermediates formed along each pathway.
Topics: Animals; Chickens; Muramidase; Particle Size; Peptide Mapping; Protein Conformation; Surface Properties
PubMed: 24089713
DOI: 10.1063/1.4811343 -
Physical Chemistry Chemical Physics :... Sep 2013Single molecule bioelectronic circuits provide an opportunity to study chemical kinetics and kinetic variability with bond-by-bond resolution. To demonstrate this...
Single molecule bioelectronic circuits provide an opportunity to study chemical kinetics and kinetic variability with bond-by-bond resolution. To demonstrate this approach, we examined the catalytic activity of T4 lysozyme processing peptidoglycan substrates. Monitoring a single lysozyme molecule through changes in a circuit's conductance helped elucidate unexplored and previously invisible aspects of lysozyme's catalytic mechanism and demonstrated lysozyme to be a processive enzyme governed by 9 independent time constants. The variation of each time constant with pH or substrate crosslinking provided different insights into catalytic activity and dynamic disorder. Overall, ten lysozyme variants were synthesized and tested in single molecule circuits to dissect the transduction of chemical activity into electronic signals. Measurements show that a single amino acid with the appropriate properties is sufficient for good signal generation, proving that the single molecule circuit technique can be easily extended to other proteins.
Topics: Biocatalysis; Kinetics; Models, Molecular; Muramidase
PubMed: 23752924
DOI: 10.1039/c3cp51356d -
The Biochemical Journal Jun 2004Lysozyme is one of the most abundant antimicrobial proteins in the airspaces of the lung. Mice express two lysozyme genes, lysozyme M and P, but only the M enzyme is... (Comparative Study)
Comparative Study
Lysozyme is one of the most abundant antimicrobial proteins in the airspaces of the lung. Mice express two lysozyme genes, lysozyme M and P, but only the M enzyme is detected in abundance in lung tissues. Disruption of the lysozyme M locus significantly increased bacterial burden and mortality following intratracheal infection with a Gram-negative bacterium. Unexpectedly, significant lysozyme enzyme activity (muramidase activity) was detected in the airspaces of uninfected lysozyme M-/- mice, amounting to 25% of the activity in wild-type mice. Muramidase activity in lysozyme M-/- mice was associated with increased lysozyme P mRNA and protein in lung tissue and bronchoalveolar lavage fluid respectively. The muramidase activity of recombinant lysozyme P was less than that of recombinant M lysozyme. Recombinant P lysozyme was also less effective in killing selected Gram-negative bacteria, requiring higher concentrations than lysozyme M to achieve the same level of killing. The lower antimicrobial activity of P lysozyme, coupled with incomplete compensation by P lysozyme in lysozyme M-/- mice, probably accounts for the increased susceptibility of null mice to infection. Recombinant lysozyme M and P were equally effective in killing selected Gram-positive organisms. This outcome suggests that disruption of both M and P loci would significantly increase susceptibility to airway infections, particularly those associated with colonization by Gram-positive organisms.
Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Candida albicans; Cell Line; Escherichia coli; Humans; Insecta; Klebsiella pneumoniae; Mice; Mice, Knockout; Microbial Sensitivity Tests; Micrococcus; Molecular Sequence Data; Muramidase; Pseudomonas aeruginosa; Staphylococcus aureus; Streptococcus agalactiae
PubMed: 14977423
DOI: 10.1042/BJ20031810 -
The Journal of Physical Chemistry. B Nov 2013Circular permutations usually retain the native structure and function of a protein while inevitably perturbing its folding dynamics. By using simulations with a...
Circular permutations usually retain the native structure and function of a protein while inevitably perturbing its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories, we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, although subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, in which one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed.
Topics: Bacteriophage T4; Muramidase; Optical Tweezers; Protein Folding; Protein Structure, Tertiary; Thermodynamics
PubMed: 24090448
DOI: 10.1021/jp406818t -
Protein Science : a Publication of the... Mar 2018The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O gas-pressure NMR spectroscopy and molecular...
The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O in solvent to 8.9 mM, O -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O binding sites and their atomic coordinates from the 1/r distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O revealed dioxygen ingress and egress as well as rotational and translational motions of O in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O association with the internal protein cavities.
Topics: Binding Sites; Crystallography, X-Ray; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Dynamics Simulation; Muramidase; Oxygen; Protein Conformation; Protein Domains; Xenon
PubMed: 29271012
DOI: 10.1002/pro.3371 -
Scientific Reports Nov 2017Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein...
Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.
Topics: Animals; Calorimetry; Chick Embryo; Kinetics; Muramidase; Protein Engineering; Protein Unfolding
PubMed: 29176711
DOI: 10.1038/s41598-017-16360-y